Inhibition of neovascularization by inhibition of prostanoid IP receptors

ABSTRACT

There are provided inter alia methods and compounds useful for decreasing choroidal neovascularization in a subject in need thereof.

CROSS REFERENCE TO RELATION APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/778,055, filed Mar. 12, 2013, the entiredisclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Neovascularization refers generally to the formation of functionalvascular (e.g., microvascular) networks. The formation may involveproliferation of blood vessels in tissue not normally containing bloodvessels, or of blood vessels of a different type than found in theneovascularized tissue under normal physiological conditions. Forexample, choroidal neovascularization (CNV) involves the invasion of newblood vessels at the choroid. CNV can involve abnormalities in Bruch'smembrane forming the innermost layer of the choroid. The resultingneovascular tissue can cause physical separation of the laminarstructure of the retina and destruction of normal retinal tissue in turncausing leakage, bleeding, and blindness. Accordingly, CNV is associatedwith a variety of diseases of the eye including age-related maculardegeneration (AMD). For example, in the so-called “wet” form of AMD, CNVdevelops in the choriocapillaris and subretinal space, disrupting theretinal pigment epithelium (RPE) which results in significant loss ofthe central vision. See e.g., Green, 1999, Mol. Vis. 5:27. Indeed, AMDis a leading cause of blindness in older populations of developedcountries. See e.g., Resnikoff et al., 2004, Bull. World Health Organ.82:844-851.

Occlusion of retinal vessels leading to ischemia and/or hypoxia iscommon in CNV. See e.g., Michaelson I., 1948, Trans Ophthalmol Soc UK.68:137-180; Ashton N., 1957, Am J. Ophthalmol. 44:7-17; 6. Shimizu K, etal., 1981, Ophthalmology 88:601-612. Extensive evidence on theinvolvement of Vascular Endothelial Growth Factor (VEGF) has beenprovided. For example, VEGF is upregulated in hypoxia, and levels ofVEGF are increased with ischemic retinopathy. See e.g., Shweiki D, etal., 1992, Nature 359:843-845; Plate K H, et al., 1992, Nature359:845-848; Forsythe J A, et al., 1996, Mol Cell Biol. 16:4604-4613;Adamis A P, et al., 1994, Am J Ophthalmol 118:445-450; 12. Aiello L P,et al., 1994, N Engl J Med. 331:1480-1487; Malecaze F, et al., 1992,Arch Ophthalmol. 112:1476-1482; Pe'er J, et al., 1995, Lab Invest.72:638-645; Miller J W, et al., 1994, Am J Pathol. 145:574-584; Pierce EA, et al., 1995, Proc Natl Acad Sci USA. 92:905-909. Indeed, increasedexpression of VEGF in retinal photoreceptors of transgenic micestimulates neovascularization within the retina, and VEGF antagonistspartially inhibit retinal neovascularization in animal models. See e.g.,Okamoto N, et al. 1997, Am J Pathol. 151:281-291; To be T, et al., 1998,Invest Ophthalmol V is Sci. 39:180-188; Aiello L P, et al., 1995, ProcNatl Acad Sci USA. 92:10457-104611 Robinson G S, et al., 1996, Proc NatlAcad Sci USA. 93:4851-4856; Adamis A P, et al., 1996, Arch Ophthalmol.114:66-71. Moreover, overexpression of VEGF in the subretinal spaceresults in neovascularization of the choroidal membranes and death ofthe overlying photoreceptor cells. See e.g., Spilsbury, K., et al.,2000, Am. J. Pathology 157:135-144.

Accordingly, there is a need to decrease neovascularization (e.g., CNV)in subjects in need thereof. There are provided herein methods andcompounds useful, inter alia, to decrease neovascularization.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, there is provided a method for decreasingneovascularization in a subject in need thereof. The method includesadministering to the subject an effective amount of an IP receptorantagonist, thereby decreasing the neovascularization.

In another aspect, there is provided a pharmaceutical compositionincluding an IP receptor antagonist and a pharmaceutically acceptableexcipient.

In another aspect, there is provided an ophthalmic pharmaceuticalformulation including an IP receptor antagonist and an ophthalmicpharmaceutically acceptable excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: IP receptor agonist Cicaprost dose dependently induces VEGFsecretion in TNFα-stimulated human macrophages. ***p<0.01, ***p<0.001compared to the vehicle control. Average EC50=2.3 nM, n=4 donors. FIG.1B: IP receptor antagonist Formula I dose dependently inhibits 100 nMCicaprost-induced VEGF secretion in TNFα-stimulated human macrophagesfrom two individual donors. ***p<0.001 compared to Cicaprost control.

FIG. 2 depicts that blockade of IP receptors alone is sufficient toinhibit laser-induced choroidal neovascularization (CNV). FIG. 2A: Ratvs. Rat (n=6); FIG. 2B: Eye vs. Eye (n=12); FIG. 2C: Lesion vs. lesion(n=38). Vehicle is 50% propylene glycol in H₂0. IP antagonist is FormulaII.

DETAILED DESCRIPTION OF THE INVENTION I. Methods

In a first aspect, there is provided a method for decreasingneovascularization in a subject in need thereof. The method includesadministering to the subject an effective amount of an IP receptorantagonist, thereby decreasing the neovascularization.

The terms “subject,” “patient” and the like refer, except whereindicated otherwise, to mammals such as humans and non-human primates,as well as rabbits, rats, mice, goats, pigs, and other mammalianspecies. The term does not necessarily indicate that the subject hasbeen diagnosed with a particular disease but typically refers to anindividual under medical supervision. In one embodiment, the subject isa human. The phrases “subject in need” and the like refer to a subjecthaving a perceived or diagnosed disease or condition (e.g., age-relatedmacular degeneration).

The term “neovascularization” refers in the usual and customary sense tothe formation of vasculature, such as de novo formation of functionalmicrovascular networks having red blood cell perfusion. In oneembodiment, neovascularization is of the choroid layer of the eye.Accordingly, the terms “choroidal neovascularization,” “CNV” and thelike refer to the formation of vasculature in the choroid, such asfunctional microvascular networks having red blood cell perfusion withinor associated with the choroid. Thus, in one embodiment, there isprovided a method for decreasing choroidal neovascularization in asubject in need thereof. The method includes administering to thesubject an effective amount of an IP receptor antagonist, therebydecreasing the neovascularization.

The terms “IP receptor,” “prostaglandin I₂ receptor,” “prostacyclinreceptor,” “PTGIR” and the like refer in the usual and customary senseto a receptor for prostacyclin (i.e., PGI₂, prostaglandin I₂). Thenatural receptors are members of the G protein-coupled receptor genesuperfamily. Upon activation, an IP receptor typically causes anelevation in intracellular cAMP via direct stimulation of adenylatecyclase. Human IP receptors are well-known in the art and are discussedin detail, for example, in Boie et al., 1994, J. Biol. Chem.269:12173-12178.

Useful IP receptor antagonists include, for example, those set forth in:EP 0 902 018 A2 (e.g. 2-(arylphenyl)amino-imidazoline derivatives whichare IP receptor antagonists); U.S. Pat. No. 6,184,242 (e.g.2-(substituted-phenyl)amino-imidazoline derivatives); WO 02/070514 (e.g.alkoxycarbonylamino heteroaryl carboxylic acid derivatives); WO02/070500 (e.g. alkoxycarbonylamino benzoic acid or alkoxycarbonylaminotetrazolyl phenyl derivatives); WO 02/40453 (e.g. substituted2-phenylaminoimidazoline phenyl ketone derivatives); WO 01/68591 (e.g.carboxylic acid derivatives); U.S. Pat. No. 6,417,186; U.S. Pat. No.6,569,860; U.S. Pat. No. 6,903,086; U.S. Pat. No. 6,693,098; U.S. Pat.No. 6,998,414 and U.S. Pat. No. 7,056,903.

Additional IP receptor antagonists for use in the methods set forthherein are as follows. The structure of selexipag(2-(4-((5,6-diphenylpyrazin-2-yl)(isopropyl)amino)butoxy)-N-(methylsulfonyl)acetamide) is:

and includes pharmaceutically acceptable salts of the above. Thestructure of 2-(3-(4,5-diphenyl-[2,4′-bioxazol]-5′-yl)phenoxy)aceticacid is:

and includes pharmaceutically acceptable salts of the above. Thestructure of3-(4-fluorophenyl)-2-(5-(4-fluorophenyl)benzofuran-2-yl)(methoxycarbonyl)amino)propanoicacid is:

and includes pharmaceutically acceptable salts of the above. Thestructure of(R)-3-phenyl-2-((((5-phenylbenzofuran-2-yl)methoxy)carbonyl)amino)propanoicacid is:

and includes pharmaceutically acceptable salts of the above.

Other useful IP receptor antagonists include the compounds of Formulas Iand II below. The structure of Formula I is:

and includes pharmaceutically acceptable salts of the above. Thestructure of Formula II is:

and includes pharmaceutically acceptable salts of the above.

An example of an IP receptor agonist useful in the experimental workdescribed herein is cicaprost. The structure of cicaprost is:

and includes pharmaceutically acceptable salts of the above.

In one embodiment, neovascularization (e.g. choroidalneovascularization) is associated with excess levels of vascularendothelial growth factor (VEGF). The terms “excess levels” and the likein the context of concentrations of biological molecules (e.g., VEGF)refer to concentrations which are greater than concentrations foundunder normal physiological conditions (e.g., in the absence ofneovascularization). Methods for determining the concentrations ofbiological molecules in the subject are well known in the art andinclude, e.g., biological assays, direct quantification ofconcentrations, and the like. Accordingly, the phrase“neovascularization is associated with excess levels of VEGF” and thelike refer to neovascularization wherein the concentration of VEGF inthe subject is greater than the concentration found in the absence ofneovascularization. The greater concentration of VEGF can be at the siteof neovascularization or in the tissue surrounding the site ofneovascularization or associated with it.

The terms “vascular endothelial growth factor,” “VEGF” and the likerefer in the usual and customary sense to a sub-family of theplatelet-derived growth factor family, which is a member of thecystine-knot family of growth factors. VEGF's include, for example,subtypes such as VEGF-A, VEGF-B, VEGF-C, VEGF-D, and the like. VEGF is asignal protein that typically stimulates vasculogenesis and angiogenesisand can contribute to disease when overexpressed. For example,overexpression of VEGF can cause vascular disease in the retina of theeye and other parts of the body. Drugs such as bevacizumab (humanizedmonoclonal antibody to VEGF-A) can inhibit VEGF and control or slowthose diseases.

In one embodiment, administration of an effective amount of the IPreceptor antagonist inhibits (e.g. decreases) the secretion of VEGF. Inone embodiment, the secretion of VEGF is inhibited by 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or even greater, with respect to thesecretion of VEGF in the absence of administration of the IP receptorantagonist. Thus, in one embodiment, the concentration of VEGF isdecreased due to the decrease in secretion of VEGF.

In one embodiment, the neovascularization is choroidalneovascularization (CNV). Accordingly, the phrases “choroidalneovascularization is associated with excess levels of VEGF” and thelike refer to choroidal neovascularization wherein the concentration ofVEGF in the subject is greater than the concentration found in theabsence of choroidal neovascularization. The greater concentration ofVEGF can be at the site of choroidal neovascularization or in the tissuesurrounding the site of choroidal neovascularization or associated withit.

In one embodiment, the choroidal neovascularization is associated withage-related macular degeneration (e.g., wet type age-related maculardegeneration). The terms “age-related macular degeneration,” “AMD” andthe like refer to a medical condition, typically afflicting older adults(e.g., >=50-yrs age), which results in loss of vision, especially in themacular visual field. The condition typically results from damage to theretina. In the “dry” (nonexudative) form of AMD, cellular debris (i.e.,drusen) accumulates between the retina and the choroid, and the retinacan become detached. In the more severe “wet” (exudative) form, bloodvessels emerge from the choroid in the choriocapillaris through Bruch'smembrane. This can ultimately lead to blood and protein leakage belowthe macula. Bleeding, leaking and scarring from these blood vessels caneventually cause irreversible damage to the photoreceptors. Moreover,the retina can become detached.

In one embodiment, the CNV is induced by exposure to a laser. In oneembodiment, the IP receptor antagonist is capable of reducing VEGFlevels (e.g., reducing VEGF levels in the choroid or in tissueassociated with the choroid).

In one embodiment, the IP receptor antagonist has the structure ofFormula (II), and the neovascularization is associated with excesslevels of VEGF. In one embodiment, the neovascularization is choroidalneovascularization. In one embodiment, the choroidal neovascularizationis associated with wet-type age-related macular degeneration. In oneembodiment, the choroidal neovascularization is laser-induced.

II. Pharmaceutical Compositions

In another aspect, there is provided a pharmaceutical compositionincluding an IP receptor antagonist in combination with apharmaceutically acceptable excipient (e.g., carrier). In oneembodiment, the pharmaceutical composition is an ophthalmicpharmaceutical composition. The term “ophthalmic pharmaceuticalcomposition” refers to a pharmaceutical composition suitable foradministration to the eye. The “ophthalmic pharmaceutical composition”may be formulated for delivery of an IP receptor antagonist to thechoroid of a subject.

The phrases “pharmaceutically acceptable excipient,” “pharmaceuticallyacceptable carrier” and the like as used herein refer to pharmaceuticalexcipients, e.g., pharmaceutically, physiologically, acceptable organicor inorganic carrier substances suitable for administration, and whichdo not deleteriously react with the active agent. In one embodiment, apharmaceutical composition can be sterilized and/or mixed with auxiliaryagents such as lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,and/or aromatic substances and the like that do not deleteriously reactwith the compounds disclosed herein. The term “ophthalmicpharmaceutically acceptable excipient” refers to a pharmaceuticallyacceptable excipient suitable for administration to the eye.

The term “pharmaceutically acceptable salt” is meant to include salts ofthe active compounds that are prepared with relatively nontoxic acids orbases, depending on the particular substituents found on the compoundsdescribed herein. When compounds disclosed herein contain relativelyacidic functionalities, base addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds disclosed herein contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, oxalic,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galacturonic acids and the like (see, for example, Bergeet al., Journal of Pharmaceutical Science, 1977, 66, 1-19). Certainspecific compounds disclosed herein contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

The compounds disclosed herein may exist as salts, such as withpharmaceutically acceptable acids. Examples of such salts includehydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates,maleates, acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates,(−)-tartrates, or mixtures thereof including racemic mixtures),succinates, benzoates, and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in theart.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

In addition to salt forms, embodiments disclosed herein providecompounds in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide certain of the compounds disclosedherein. Additionally, prodrugs can be converted to certain compoundsdisclosed herein by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to certaincompounds disclosed herein when placed in a transdermal patch reservoirwith a suitable enzyme or chemical reagent.

Certain compounds disclosed herein can exist in unsolvated forms as wellas solvated forms, including hydrated forms. In general, the solvatedforms are equivalent to unsolvated forms and are encompassed within thescope of the present invention. Certain compounds disclosed herein mayexist in multiple crystalline or amorphous forms. In general, allphysical forms are equivalent for the uses contemplated by the presentinvention and are intended to be within the scope of the presentinvention.

Certain compounds disclosed herein possess asymmetric carbon atoms(optical centers) or double bonds; the racemates, diastereomers,tautomers, geometric isomers, and individual isomers are encompassedwithin the scope of the present invention. The compounds disclosedherein do not include those that are known in the art to be too unstableto synthesize and/or isolate.

The compounds disclosed herein may also contain unnatural proportions ofatomic isotopes at one or more of the atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I), or carbon-14 (¹⁴C). All isotopic variations of the compoundsdisclosed herein, whether radioactive or not, are encompassed within thescope of the present invention.

In one embodiment, the pharmaceutical composition includes an IPreceptor antagonist at a concentration in the range of about 0.01% to1.00% (w/v). In one embodiment, the concentration of the IP receptorantagonist is about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%,0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%,0.18%, 0.19%, 0.20%, 0.30%, 0.40%, 0.50%, 0.60%, 0.70%, 0.80%, 0.90%, or1.00% (w/v), or even greater.

A. Formulations

The compounds disclosed herein can be prepared and administered in awide variety of ophthalmic, oral, parenteral, and topical dosageformulations. Thus, the compounds disclosed herein can be administeredby injection (e.g. intravenously, intramuscularly, intracutaneously,subcutaneously, intraduodenally, or intraperitoneally). Also, thecompounds described herein can be administered by inhalation, forexample, intranasally. Additionally, the compounds disclosed herein canbe administered transdermally or by ocular instillation. Multiple routesof administration (e.g., intramuscular, oral, transdermal, ocularinstillation) are contemplated that can be used to administer thecompounds disclosed herein. Accordingly, embodiments of the presentinvention also provide pharmaceutical compositions (e.g., an ophthalmicpharmaceutical composition) which include a pharmaceutically acceptablecarrier or excipient and one or more compounds (e.g., IP receptorantagonist).

In another aspect, there is provided an ophthalmic pharmaceuticalformulation. An “ophthalmic pharmaceutical formulation” includes anophthalmic pharmaceutical composition and is formulated to beadministered to the eye. In one embodiment, the ophthalmicpharmaceutical formulation is instilled in the eye. In one embodiment,the ophthalmic pharmaceutical formulation is injected into the eye, orinto tissue surrounding or associated with the eye.

In one embodiment, the ophthalmic pharmaceutical formulation includes anIP receptor antagonist at a concentration in the range of about 0.01% to1.00% (w/v). In one embodiment, the concentration of the IP receptorantagonist is about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%,0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%,0.18%, 0.19%, 0.20%, 0.30%, 0.40%, 0.50%, 0.60%, 0.70%, 0.80%, 0.90%, or1.00% (w/v), or even greater.

The ophthalmic pharmaceutical formulation can include buffers to adjustthe pH to a desirable range for ophthalmic use. Generally, a pH ofaround 6-8 is desired, and in certain ophthalmic pharmaceuticalformulations a pH of 7.4 is desired. Many buffers including salts ofinorganic acids such as phosphate, borate, and sulfate are known. In oneembodiment, the buffer maintains the pH of the ophthalmic pharmaceuticalformulation in the range of about 6.5 to 7.5. In one embodiment, thebuffer maintains the pH of the ophthalmic pharmaceutical formulation inthe range of about 7.0 to 7.4.

In one embodiment, the ophthalmic pharmaceutical formulation includesone or more viscosity-enhancing agents, or thickening agents. Thickeningagents are used for a variety of reasons, ranging from improving theform of the formulation for convenient administration to improving thecontact with the eye to improve bioavailability. The viscosity-enhancingagent may comprise a polymer containing hydrophilic groups such asmonosaccharides, polysaccharides, ethylene oxide groups, hydroxylgroups, carboxylic acids or other charged functional groups. While notintending to limit the scope of the invention, some examples of usefulviscosity-enhancing agents are sodium carboxymethylcellulose,hydroxypropyl methylcellulose, povidone, polyvinyl alcohol, andpolyethylene glycol. In one embodiment, viscosity-enhancing agents areemployed at a level between about 0.01% and about 2% (w/v).

In one embodiment, the ophthalmic pharmaceutical formulation includesone or more tonicity agents useful to adjust the ophthalmicpharmaceutical formulation to the desired isotonic range. Tonicityagents are well known in the art and some examples include glycerin,mannitol, sorbitol, sodium chloride, and other electrolytes. In oneembodiment, the concentration of tonicity agent is in the range of about0.1 to 2.00% (w/v). In one embodiment, the concentration of tonicityagent is in the range of about 1.15 to 1.30% (w/v). In one embodiment,the concentration of tonicity agent is about 0.10%, 0.20%, 0.30%, 0.40%,0.50%, 0.60%, 0.70%, 0.80%, 0.90%, 1.00%, 1.10%, 1.20%, 1.30%, 1.40%,1.50%, 1.60%, 1.70%, 1.80%, 1.90%, or 2.00%. In one embodiment, theconcentration of tonicity agent is about 1.15%, 1.16%, 1.17%, 1.18%,1.19%, 1.20%, 1.21%, 1.22%, 1.23%, 1.24%, 1.25%, 1.26%, 1.27%, 1.28%,1.29%, or 1.30% (w/v).

In one embodiment, the ophthalmic pharmaceutical formulation includes asolubilizer (e.g., surfactant or other appropriate co-solvent) in orderto facilitate solubilization of one or more components of the ophthalmicpharmaceutical formulation. Such solubilizers include Polysorbate 20,60, and 80, Pluronic F-68, F-84, and P-103, cyclodextrin,hydroxy-beta-cyclodextrin, solutol, polyoxyethylene 40 stearate, andpolyoxyl 35 castor oil. Such solubilizers can be employed at a levelbetween about 0.01% and about 2% by weight. In one embodiment, thesolubilizer is present in the range of about 0.01% to 0.20% (w/v). Inone embodiment, the solubilizer is present at 0.01%, 0.02%, 0.03%,0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%,0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, or 0.20% (w/v). In oneembodiment, the solubilizer is polysorbate 80.

In one embodiment, the ophthalmic pharmaceutical formulation includes apreservative. In one embodiment, the preservative is benzalkoniumchloride, chlorine dioxide, chlorobutanol, thimerosal, phenylmercuricacetate, or phenylmercuric nitrate. In one embodiment, the preservativeis present at a concentration in the range of about 0.01% to 0.05%(w/v). In one embodiment, the preservative is present at a concentrationin the range of about 0.015% to 0.025% (w/v). In one embodiment, theconcentration of the preservative is about 0.015%, 0.016%, 0.017%,0.018%, 0.019%, 0.020%, 0.021%, 0.022%, 0.023%, 0.024%, or 0.025% (w/v).In one embodiment, the preservative is benzalkonium chloride.

The compositions disclosed herein may additionally include components toprovide sustained release and/or comfort. Such components include highmolecular weight, anionic mucomimetic polymers, gelling polysaccharides,and finely-divided drug carrier substrates. These components arediscussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841;5,212,162; and 4,861,760, the entire contents of each of which areincorporated herein by reference in their entirety and for all purposes.

B. Effective Dosages

Pharmaceutical compositions contemplated herein include compositionswherein the active ingredient is contained in an effective amount, i.e.,in an amount effective to achieve its intended purpose. An “effectiveamount” is an amount sufficient to accomplish a stated purpose (e.g.,achieve the effect for which it is administered, treat a disease, reduceone or more symptoms of a disease or condition). An example of an“effective amount” is an amount sufficient to contribute to thetreatment, prevention, or reduction of a symptom or symptoms of adisease, which can be referred to as a “therapeutically effectiveamount.” A “reduction” of a symptom or symptoms (and grammaticalequivalents of this phrase) means decreasing of the severity orfrequency of the symptom(s), or elimination of the symptom(s). Theactual amount effective for a particular application will depend, interalia, on the condition being treated. For example, when administered inmethods to treat neovascularization (e.g., choroidalneovascularization), such compositions will contain amounts of activeingredients effective to achieve the desired result (e.g. decreasing theextent of neovascularization in a subject).

The dosage and frequency (single or multiple doses) of compoundsadministered can vary depending upon a variety of factors, includingroute of administration; size, age, sex, health, body weight, body massindex, and diet of the recipient; nature and extent of symptoms of thedisease being treated (e.g., the disease responsive to an IP receptorantagonist); presence of other diseases or other health-relatedproblems; kind of concurrent treatment; and complications from anydisease or treatment regimen. Other therapeutic regimens or agents canbe used in conjunction with the methods and compounds disclosed herein.

For any compound described herein or combination thereof, thetherapeutically effective amounts can be initially determined from cellculture assays. Target concentrations will be those concentrations ofactive compound(s) that are capable of decreasing neovascularization(e.g., choroidal neovascularization) as measured, for example, usingmethods known in the art.

Therapeutically effective amounts for use in humans may be determinedfrom animal models. For example, a dose for humans can be formulated toachieve a concentration that has been found to be effective in animals.The dosage in humans can be adjusted by monitoring neovascularization(e.g., choroidal neovascularization) and adjusting the dosage upwards ordownwards, as described above.

Dosages may be varied depending upon the requirements of the subject andthe compound being employed. The dose administered to a subject, in thecontext of certain embodiments disclosed herein, should be sufficient toeffect a beneficial therapeutic response in the subject over time. Thesize of the dose also will be determined by the existence, nature, andextent of any adverse side effects. Generally, treatment is initiatedwith smaller dosages, which are less than the optimum dose of thecompound. Thereafter, the dosage is increased by small increments untilthe optimum effect under circumstances is reached.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered compounds effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

Utilizing the teachings provided herein, an effective therapeutictreatment regimen can be planned that does not cause substantialtoxicity and yet is entirely effective to treat the clinical symptomsdemonstrated by the particular patient. This planning should involve thecareful choice of active compound by considering factors such ascompound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration, and the toxicity profile of the selected agent.

C. Toxicity

The ratio between toxicity and therapeutic effect for a particularcompound is its therapeutic index and can be expressed as the ratiobetween LD₅₀ (the amount of compound lethal in 50% of the population)and ED₅₀ (the amount of compound effective in 50% of the population).Compounds that exhibit high therapeutic indices are preferred.Therapeutic index data obtained from cell culture assays and/or animalstudies can be used in formulating a range of dosages for use in humans.The dosage of such compounds preferably lies within a range of plasmaconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. See, e.g. Fingl etal., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch. 1, p. 1, 1975.The exact formulation, route of administration, and dosage can be chosenby the individual physician in view of the patient's condition and theparticular method in which the compound is used.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

III. Examples Example 1 Investigation of VEGF Secretion and Inhibitionin Culture

A TNFα-stimulated human macrophage system was employed, as known in theart, to assess the effect of IP receptor agonist and IP receptorantagonist on induction of VEGF secretion. As depicted in FIG. 1A,cicaprost dose dependently induces VEGF secretion in TNFα-stimulatedhuman macrophages with EC₅₀=2.3 nM.

In order to test whether a IP receptor antagonist can reverse the effectof cicaprost-induced VEGF secretion in TNFα-stimulated humanmacrophages, the IP receptor antagonist Formula I was employed. Asdepicted in FIG. 1B, Formula I dose dependently inhibits the secretionof VEGF in TNFα-stimulated human macrophages which can been exposed to100 nM cicaprost. The human macrophages had been obtained from twoseparate donors, as indicated in FIG. 1B.

Example 2 Investigation of Blockade of IP Receptors in Laser-Induced CNV

A laser-induced model of CNV was employed, as known in the art, todetermine whether blockade of IP receptors alone is sufficient toinhibit CNV. See e.g., Francois et al., 1975, Am. J. Ophthalmol.79:206-210; Edelman & Castro, 2000, Exp. Eye Res. 71:523-533; Goody, R.J., et al., 2011, Exp. Eye. Research 92:464-472.

Test rats were administered laser treatment after which vehicle (50%propylene glycol in H₂O) or IP receptor antagonist Formula II wasadministered. The area of subsequently identified CNV was quantified bymethods known in the art. As shown in FIG. 2A, the area (μm²) of CNV wassignificantly reduced per test subject after administration of IPreceptor antagonist. As depicted in FIG. 2B, the area of CNV was alsoreduced as judged by a comparison of each eye. Moreover, as depicted inFIG. 2C, the area of CNV was reduced on a per lesion basis.

What is claimed is:
 1. A method for decreasing neovascularization in asubject in need thereof, comprising administering to said subject aneffective amount of an IP receptor antagonist, thereby decreasing saidneovascularization.
 2. The method of claim 1, wherein saidneovascularization is associated with excess levels of vascularendothelial growth factor (VEGF).
 3. The method of claim 2, wherein saidIP receptor antagonist inhibits secretion of VEGF in said subject. 4.The method of claim 3, wherein said neovascularization is choroidalneovascularization.
 5. The method of claim 4, wherein said choroidalneovascularization is associated with wet type age-related maculardegeneration.
 6. The method of claim 4, wherein said choroidalneovascularization due to physical or chemical trauma or a secondaryresult of cardiovascular disease.
 7. The method of claim 1, wherein saidIP receptor antagonist has the structure of Formula II:


8. The method of claim 7, wherein said choroidal neovascularization isassociated with excess levels of vascular endothelial growth factor(VEGF).
 9. The method of claim 8, wherein said IP receptor antagonistinhibits secretion of VEGF in said subject.
 10. The method of claim 7,wherein said neovascularization is choroidal neovascularization.
 11. Themethod of claim 10, wherein said choroidal neovascularization isassociated with wet type age-related macular degeneration.
 12. Themethod of claim 10, wherein said choroidal neovascularization is laserinduced.